Project Summary Bladder cancer represents a major public health burden. The cost per bladder cancer patient from diagnosis to death is the highest among all cancers ($96,000 to $187, 000 per patient in the US). Our long-term goal is to develop new chemopreventive agents to reduce the burden of bladder cancer. Because exposure to carcinogens is the major risk factor for bladder cancer and many potentially protective compounds contained in plant-derived food are concentrated in the urine, it is plausible that bioactive agents in plants may play a role in bladder cancer chemoprevention. Flavokawain A is the predominant chalcone identified from kava root extract. The promise of flavokawain A is based on the following data : 1) an epidemiological study reported that high kava consumption correlated with lower incidences of cancers despite the presence of many smokers in the populations of the South Pacific Islands;2) In vitro cell culture studies showed that flavokawains but not kavalactones in kava extracts exhibited strong antiproliferative and apoptotic effects on human bladder cancer cells characterized as low- and high- grades;3) Flavokawain A inhibited tumor growth in xenograft models of superficial and invasive bladder cancer without any noticeable side effects;and 4) The chemical structure of flavokawain A, similar to some other chalcones, leads us to predict that it would cause no direct damage to DNA, bind to beta-tubulin, and induce phase II enzymes to protect against carcinogenesis. We hypothesize flavokawain A has chemopreventive effects on bladder epithelium via enhancing cell cycle regulation and increasing the sensitivity of apoptotic mechanisms. Three mouse models will be used to test this hypothesis: a carcinogen [4-hydroxybutyl (butyl) nitrosamine (OH-BBN)]-induced carcinogenesis model in mice bladders, and two spontaneous mouse bladder transgenic carcinogenesis models, each of which will address distinct issues. The OH-BBN model in mice will be used to examine flavokawain A's utility in primary prevention;for preventing the first occurrence of bladder cancer in those with risk factors such as cigarette smoking and industrial exposures. The transgenic models each induce a separate precursor state to advanced bladder cancer: either papillary tumors or carcinoma in situ, as seen in early stages of clinical bladder cancer. Examining flavokawain A in these transgenic models will provide information about its usefulness in secondary prevention, which clinically will address preventing bladder cancer recurrence and progression. Our microarray analysis has identified a few potential transcriptional targets (i.e. BIM, BID, survivin, Plk1, 14-3-3gamma, and SKP-2) for flavokawain A when inducing apoptosis and cell cycle arrests. We will validate these targets by examining their expression in vivo in these tested animal models and by using RNA interference and overexpression vectors to determine their contributions to flavokawain A's action in vitro, as well as by studying the down-stream events of these targets. Together, these studies will provide a firm answer to the promise of flavokawin A as a chemoprventative and/or chemotherapeutic agent.